Our goal is to understand the mechanisms of loss of heterozygosity (LOH). LOH results from transfer of information from one homologous chromosome to the other. LOH occurs frequently in tumors but is rarely evident in normal cells. LOH events can alter DNA sequence over many megabases, spanning many genes, perturbing critical pathways by altering gene activities and expression levels. Thus, even though LOH occurs much less frequently than point mutation, LOH contributes profoundly to tumor biology. LOH depends upon homology-directed repair (HDR) pathways, and it was long presumed to depend exclusively upon HDR acting at DNA DSBs. However our recent results challenge that view. We have discovered an alternative pathway of HDR, which is stimulated upon downregulation of canonical HDR and supports transfer of information from a nicked donor DNA to a nicked recipient (Davis and Maizels, PNAS, in press). The mechanisms of LOH have not been clearly defined, and this limits our ability to minimize this mutagenic signature as a source of genomic instability. We propose to define the mechanisms of LOH, and establish how LOH is activated in human tumors. To do this we propose the following two Specific Aims: Aim 1. We will define the factors that carry out LOH. Aim 2. We will determine how LOH is regulated in human cancer cells Significance: Therapies that target the factors that promote or regulate LOH may diminish LOH frequencies, and reduce the intratumor heterogeneity that compromises many therapies. Relevance Loss of heterozygosity (LOH) occurs frequently in tumors, where it contributes to driver mutations that promote development of cancer and intratumor heterogeneity that thwarts therapy. Our goal is to understand the mechanism of LOH. This will make it possible to downregulate LOH frequencies and diminish the contribution of LOH to tumorigenesis.